PROJECT SUMMARY Transcription in eukaryotes is initiated following the coordinated action of dozens to hundreds of proteins. These activities result in the recruitment of RNA Polymerase II (Pol II) to core promoters and the subsequent initiation process. During initiation, Pol II must be directed to the transcription start site (TSS) followed by TSS recognition, initial phosphodiester bond formation, and promoter clearance. The mechanism by which Pol II initiation engages the TSS and how this process determines overall promoter output is surprisingly poorly understood. Most promoters in eukaryotes initiate transcription in a broad or dispersed fashion where multiple TSSs are used. With a collaborative team in place, we wish to determine the mechanism by which Pol II engages TSSs in initiation and how initiation is affected by promoter architecture. In budding yeast, Pol II scans for TSSs and this allows us to rationalize how biochemical activities in initiation and promoter architecture may determine initiation levels. We have rationalized initiation by a model we call the Shooting Gallery that relates initiation probability to the cooperation between the Pol II active site and the activity of TFIIH, the putative motor that drives scanning. We have designed genetic, genomic, and biochemical experiments to explore and test the Shooting Gallery model. Our approaches include quantitative modeling of initiation at all yeast promoters to determine how they might be unique or what promoter features control expression levels. We will establish Schizosaccharomyces pombe and Drosophila melanogaster as models for direct tests of the conservation of promoter scanning as a mechanism for Pol II initiation. Our experiments will reveal whether there are universal initiation mechanisms in eukaryotes or not. We expect to greatly increase our power to predict promoter output based on biochemical properties of Pol II and TFIIH, our understanding of promoter architecture, and their interplay with underlying sequence, each of which are fundamental features of gene expression in all eukaryotes.